WO2008014550A1 - Marqueurs de pigmentation - Google Patents

Marqueurs de pigmentation Download PDF

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Publication number
WO2008014550A1
WO2008014550A1 PCT/AU2007/001070 AU2007001070W WO2008014550A1 WO 2008014550 A1 WO2008014550 A1 WO 2008014550A1 AU 2007001070 W AU2007001070 W AU 2007001070W WO 2008014550 A1 WO2008014550 A1 WO 2008014550A1
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WIPO (PCT)
Prior art keywords
animal
genetic marker
pigmentation
tyrosinase
marker
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PCT/AU2007/001070
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English (en)
Inventor
Herman Raadsma
Karen Fullard
Jamie Hernan Gongora Romero
Imke Tammen
Colin Cavanagh
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The University Of Sydney
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Priority claimed from AU2006904111A external-priority patent/AU2006904111A0/en
Application filed by The University Of Sydney filed Critical The University Of Sydney
Priority to AU2007281024A priority Critical patent/AU2007281024B2/en
Publication of WO2008014550A1 publication Critical patent/WO2008014550A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • the present invention relates to genetic markers for pigmentation in skin and fleece areas of animals.
  • the present invention relates to methods, systems and kits for predicting pigmentation and pigmented fibre contamination in animals using genetic markers, to associated methods, systems and kits for selecting animals using marker assisted selection, and to associated methods and systems for animal breeding and prediction of lifetime performance.
  • the present invention is predicated on the development of a genetic marker shown to be associated with pigmentation and pigmented fibre contamination in animals.
  • a method for predicting pigmentation in an animal comprising analysing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase- related protein gene, wherein said genetic marker is predictive of pigmentation in said animal.
  • the pigmentation may include pigmented skin or dark fibre contamination.
  • the tyrosinase-related protein may be TYRP-I .
  • the presence or absence of the polymorphism may be analysed through the use of nucleic acid sequences flanking the polymorphism, such as PCR primers.
  • nucleic acid sequences flanking the polymorphism such as PCR primers.
  • a method for selecting an animal using marker assisted selection comprising:
  • a system for predicting pigmentation in an animal comprising: (a) means for analyzing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein said genetic marker is predictive of pigmentation in said animal.
  • a system for selecting an animal using marker assisted selection comprising: (a) means for analyzing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein the genetic marker is predictive of pigmentation in said animal; and
  • a system for breeding an animal using marker assisted selection comprising: (a) means for analyzing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein the genetic marker is predictive of pigmentation in said animal; and (b) means for breeding said animal based on the presence or absence of the genetic marker, and
  • (c) means for selecting progeny of said animal based on the presence or absence of the genetic marker.
  • kit for predicting pigmentation in an animal wherein said kit comprises:
  • kit for selecting an animal using marker assisted selection comprising:
  • kits for breeding an animal using marker assisted selection comprising: (a) means for analyzing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein said genetic marker is predictive of pigmentation in said animal; and
  • a polypeptide comprising a tyrosinase-related protein (TYRP).
  • TYRP tyrosinase-related protein
  • the TYRP may be TYRP-I.
  • the TYRP-I may comprise the amino acid sequence set forth in SEQ ID NOS: 36, 39, 42, 45, 47, 74, 77 and 80.
  • a polynucleotide encoding the polypeptide of the eleventh aspect.
  • a vector comprising the polynucleotide of the twelfth aspect.
  • a host cell transformed with the vector of the thirteenth aspect.
  • an animal product derived from the animal of the fifteenth aspect is provided.
  • the animal product may be selected from the group comprising hair, wool, skin, cashmere, and mohair.
  • the animal product may be wool.
  • a method of sampling fibres to determine non-visual pigmentation comprises (a) genotyping a plurality of animals based on the presence or absence of at least one genetic marker in the tyrosinase-related protein gene, wherein the genetic marker is predictive of pigmentation in said animal;
  • the pigmentation is present in the form of pigmented fibres.
  • the pigmented fibres may be in the form of pigmented wool, hair, mohair or cashmere.
  • the pigmented fibres are rare and/or isolated.
  • the proportion of pigmented fibres may comprise less than
  • 0.001% of total fibre count less than 0.0001% of total fibre count, less than 0.00001% of total fibre count, less than 0.000001% of total fibre count, less than 0.0000001% of total fibre count, less than 0.00000001% of total fibre count, or less than 0.0000000001% of total fibre count.
  • QTL location for Pigmented Fibre on Chromosome 26 I. QTL location for Hoof Pigmentation on Chromosome 2; J. QTL location for Hoof Pigmentation on Chromosome 18; K. QTL location for Binary score hornsite and leg Fibre score on Chromosome 2; L. QTL location for Binary score hornsite and leg fibre score on Chromosome 8; M. QTL location for Binary score hornsite and leg fibre score on Chromosome 19.
  • FIG. 4 Black and white (top), and negative picture (bottom) of PCR products for TYRP-I exons 2, 3, 4 and 5 electrophoresed on a 2% agarose gel.
  • M marker
  • - negative control
  • C cattle
  • H human
  • M mouse
  • S sheep. Number in brackets indicates the ⁇ size of the PCR product in relation to the molecular weight of the marker.
  • Figure 5 Black and white (top), and negative photograph (bottom) of PCR products for TYRP-I exons 6, 7, 8 and 8-1 (using R2 primers described below) electrophoresed on a 2% agarose gel.
  • Figure 6. Black and white photograph of PCR products of TYRP-I exons 1 and upstream region electrophoresed on a 2% agarose gel. Primers are indicated by number according to Tables 7-9
  • Genotype '1' is significantly more associated with a higher Fibre Pigmentation Score (PigFibre) than genotype '2'.
  • FIG. 8 Parental haplotypes inherited from the Merino granddam (C7_M), and the Awassi grandsire (C7 AW), and other (partial) haplotypes. In relation thereto, animals with haplotype "34" also had a confirmed high isolated pigmented fibre count using the AWTA test.
  • Figure 9 Histogram figure of Pigmented Fibre score out of 500 for pigmented fibre, against haplotypes predictive of HIGH and LOW risk of pigmentation respectively from the 1997 mapping family
  • primer means a single-stranded oligonucleotide capable of acting as a point of initiation of template-directed DNA synthesis.
  • oligonucleotide is a single-stranded nucleic acid typically ranging in length from 2 to about 500 bases. The precise length of a primer will vary according to the particular application, but typically ranges from 15 to 30 nucleotides. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize to the template.
  • gene means the genetic constitution of an organism. This may be considered in total, or with respect to the alleles of a gene(s) or genetic marker(s).
  • homozygote refers to an organism that has identical alleles at a given locus on homologous chromosomes.
  • heterozygote refers to an organism in which different alleles are found on homologous alleles for a given locus.
  • linkage disequilibrium describes a situation in which some combinations of alleles of two or more different loci (haplotypes) occur more or less frequently within a population than would be expected by random chance alone.
  • pigmentation as used herein includes skin and fibre pigmentation, and in particular includes skin and fibre pigment contamination.
  • skin may include nose, lips, hoofs, eyelids, udder, and fibre may include hair, wool, fleece, and eyelashes.
  • the term “pigmentation” may include isolated or partial fleece colouration, also known as “white spotting", piebald, or any other form of discolouration or full- fleece colouration.
  • the term “pigmentation” may include fibres containing a degree of colouration that is not or substantially not visible to the eye.
  • dark fibre and “pigmentation” are synonymous.
  • the terms “pigment” and “pigmented” have corresponding meanings.
  • the term "genetic marker” refers to a variant or polymorphism at DNA sequence level linked to a specific chromosomal location unique to an individual's genotype, inherited in a predictable manner, and measured as a direct DNA sequence variant or polymorphism, such as at least one Single Nucleotide Polymorphism (SNP), Restriction Fragment Length Polymorphism (RFLP), or Short Tandem Repeat (STR), or as measured indirectly as a DNA sequence variant (eg. Single-strand conformation polymorphism (SSCP), Denaturing Detergent Gradient Gel Electrophoresis (DDGE).
  • SNP Single Nucleotide Polymorphism
  • RFLP Restriction Fragment Length Polymorphism
  • STR Short Tandem Repeat
  • DNA sequence variant eg. Single-strand conformation polymorphism (SSCP), Denaturing Detergent Gradient Gel Electrophoresis (DDGE).
  • single nucleotide polymorphisms or "SNP” or “SNPs”, as used herein, refers to common DNA sequence variations among subjects.
  • the DNA sequence variation is typically a single base change or point mutation resulting in genetic variation between individuals.
  • the single base change can be an insertion or deletion of a base.
  • base pair means a pair of nitrogenous bases, each in a separate nucleotide, in which each base is present on a separate strand of DNA and the bonding of these bases joins the component DNA strands.
  • a DNA molecule contains four bases; A (adenine), G (guanine), C (cytosine), and T (thymidine).
  • a and G are purine bases, typically designated by the letter “R”
  • C and T are pyrimidine bases, typically designated by the letter "Y”. Where A or T may occupy a single position it is typically designated by the letter W. Where G or C may occupy a single position it is typically designated by the letter S.
  • a or C may occupy a single position it is typically designated by the letter M.
  • G or T may occupy a single position it is typically designated by the letter K.
  • A, T or C may occupy a single position it is typically designated by the letter H.
  • G, C or T may occupy a single position it is typically designated by the letter B.
  • G, A or T may occupy a single position it is typically designated by the letter D.
  • G, C or A may occupy a single position it is typically designated by the letter V.
  • G, C, A or T may occupy a single position it is typically designated by the letter N.
  • base pair is abbreviated to "bp”
  • kilobase pair is abbreviated to kb.
  • animal refers to an individual at any stage of life, or after death, including an entity prior to birth such as a fertilised ovum, either before fusion of the male and female pro-nucleus or after the pro-nuclei have fused to form a zygote, an embryo (created by any means including somatic cell nuclear transfer) or an individual cell (N, 2N or greater); for the avoidance of doubt, this also includes a cell or a cluster of cells including stem cells and stem cell-like cells, cell line, haploid gametes and their progenitor cell lines, as well as products resulting from the gametes, including embryos.
  • chromosomes which contained genes contributing to skin and fibre pigmentation in sheep, detailed in Table 6.
  • One of the major regions with strongest significance was found on chromosome 2, and was significant for 6 skin and fibre traits.
  • mapping progeny was sufficient to account for over 50% of the differences in pigmentation, and indicated that the colour phenotype originated from the Awassi allele (Figure 7).
  • This region was shown to contain a very strong candidate gene, namely tyro sine-related protein- 1 (TYRP- 1 - Figure 3).
  • TYRP-I was sequenced by alignment of TYRP-I sequence from all known species, with conserved sequence sites used to design DNA primers for ovine sequence. A predicted ovine gene structure was then identified from comparative mapping, with ovine sequence confirming close alignment to bovine TYRP-I .
  • Genetic assay-assisted selections for animal breeding are important in that they allow selections to be made without the need for raising and phenotypic testing of progeny. In particular, such tests allow selections to occur among related individuals that do not necessarily exhibit the trait in question and that can be used in introgression strategies to select both for the trait to be introgressed and against undesirable background traits.
  • Non- limiting examples of methods for identifying markers corresponding to genetic polymorphisms between members of a population include: restriction-fragment-length polymorphism (RFLP) Bostein et al (1980) Am J Hum Genet 32:314-331; single-strand conformation polymorphism (SSCP) Fischer et al. (1983) Proc Natl Acad Sci USA 80:1579-1583, Orita et al. (1989) Genomics 5:874-879; amplified fragment-length polymorphism (AFLP) Vos et al.
  • RFLP restriction-fragment-length polymorphism
  • SSCP single-strand conformation polymorphism
  • AFLP amplified fragment-length polymorphism
  • Primers that may be used in this regard may, for example, comprise sequences set out in Tables 7, 8 and 9 and complements thereof.
  • selections may be unambiguously made based on genotypes assayed at any time after a nucleic acid sample can be collected from an individual, such as an infant animal, or even earlier in the case of testing of embryos in vitro, or testing of foetal offspring. Any source of nuclear DNA may be analyzed for scoring of genotype.
  • nucleic acids are screened that have been isolated from the blood or semen of the animal analyzed.
  • DNA from the animal to be assessed may be extracted by a number of suitable methods known to those skilled in the art. Most typically, DNA is extracted from a blood or semen sample, and in particular from peripheral blood leucocytes. A sufficient amount of cells are obtained to provide a sufficient amount of DNA for analysis, although only a minimal sample size will be needed where scoring is by amplification of nucleic acids.
  • the DNA can be isolated from the blood cells by standard nucleic acid isolation techniques known to those skilled in the art.
  • Nucleic acids used as a template for amplification may be isolated from cells, tissues or other samples according to standard methodologies. Such embodiments may find particular use with the invention, for example, in the detection of repeat length polymorphisms, such as microsatellite markers. In certain embodiments of the invention, amplification analysis is performed on whole cell or tissue homogenates or biological fluid samples without substantial purification of the template nucleic acid.
  • the amplification product may be detected or quantified.
  • the detection may be performed by visual means.
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical and/or thermal impulse signals.
  • scoring of repeat length may be performed by visual means.
  • PCR polymerase chain reaction
  • the genetic marker(s) in the tyrosinase related protein gene useful in the invention may be selected from, but is not limited to, the group of polymorphisms shown in Table 1 -0 below.
  • amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods. Amplification products isolated in this way may be eluted from an excised portion of the gel for further manipulation.
  • the nucleic acid may be removed from the excised portion of an agarose gel by heating the gel in a chao tropic solution, followed by extraction of the nucleic acid.
  • Separation and isolation of nucleic acids may also be performed by chromatographic techniques known in art. Numerous kinds of chromatography may be used in the present invention, including adsorption, partition, ion-exchange, hydroxylapatite, gel-filtration (molecular sieve), and reverse-phase. These may be performed in a number of formats including, column, paper, thin-layer, and gas chromatography as well as by batch HPLC or FPLC. In certain embodiments, the amplification products are visualized. A typical visualization method involves staining of a gel with ethidium bromide and visualization of bands under UV light.
  • a labeled nucleic acid probe is brought into contact with the amplified marker sequence.
  • the probe preferably is conjugated to a chromophore but may be radiolabeled or otherwise labeled to facilitate detection.
  • the probe is conjugated to a molecule, such as an antibody or biotin, or another molecule carrying a moiety to facilitate detection.
  • detection is by nucleic acid blotting and hybridization with a labeled probe.
  • the techniques involved in nucleic acid blotting are well known to those skilled in the art.
  • the nucleic acid blotting and hybridization may be performed under stringent conditions.
  • stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 3O 0 C for short probes (e.g., 10 to 50 nucleotides) and at least about 6O 0 C for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37°C, and a wash in 1.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37 0 C, and a wash in 0.5x to IxSSC at 55 to 5O 0 C.
  • Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37 0 C, and a wash in 0. IxSSC at 60 to 65 0 C.
  • Kits All the essential materials and/or reagents required for screening animals for genetic marker genotype in accordance with the invention may be assembled together in a kit.
  • This generally will comprise a probe or primers designed to hybridize specifically to individual nucleic acids of interest in the practice of the present invention, for example, primer sequences such as those for amplifying TYRP-I.
  • enzymes suitable for amplifying nucleic acids including various polymerases, deoxynucleotides and buffers to provide the necessary reaction mixture for amplification.
  • Such kits also may include enzymes and other reagents suitable for detection of specific nucleic acids or amplification products.
  • Such kits generally will comprise, in suitable means, distinct containers for each individual reagent or enzyme as well as for each probe or primer pair.
  • the invention also can provide for a kit which can be used to determine the TYRP-I genotype of genetic material, for example the kit may include a set of primers used for amplifying the genetic material.
  • a kit can contain a primer including a nucleotide sequence for amplifying a region of the genetic material containing one of the polymorphisms described herein.
  • the 177 offspring were male backcrossed progeny from mating between a single Awassi/Merino Fl sire (C7/A95.0453) and unselected Merino ewes, giving a progeny of 75% Merino and 25% Awassi. Animals were born from May 1997, and separated into two management cohorts according to birth dates. Table 3; Pi mentation traits and trait descri tion
  • ColourScore transformed to a binary trait
  • NoseLPig was arcsin transformed
  • LHSFibre was transformed to a binary trait.
  • a full genome scan was conducted with 204 microsatellite markers.
  • a framework map was constructed using the open source mapping programs Carthagene and Mapmaker, which was used to inform marker positions.
  • a full genome scan was conducted using a maximum likelihood analysis written in R, developed by the inventors (QTL-MLE).
  • the QTL-MLE algorithm was developed to match the particular experimental design and genotyping characteristics of the study. These included (1) incorporation of known linkage phase information resulting from the backcross design, (2) utilisation of all the relevant genotypic information, and (3) valid modelling the marker-QTL associations by means of a finite mixture model. Item (2) involved development of an algorithm to utilise information from heterozygous marker genotypes, using estimates of marker allele frequencies in the merino population to. assist this. The QTL model is fitted using a maximum likelihood routine at each putative QTL position along the length of the target chromosome (interval mapping).
  • the probability of each backcross offspring inheriting either allele from the Fl sire is provided, in terms of a posterior probability. This allows animals with a high probability of receiving the "favourable" QTL genotype to be identified.
  • a 1- LOD support interval is provided to give an indication of the precision of estimating the location of the QTL.
  • TGLAlO was found to be the closest linked mapped genetic marker and tyrosinase-related protein- 1 (TYRP-I) was identified as the most significant positional candidate gene by comparative mapping to human, cattle and rat using the Comparative Predictions of Orthologues software at the Oxford Grid Project (http://oxgrid.angis.org.au).
  • TYRP-I exons from sample C7 were amplified by PCR using the conditions described in Tables 10 and 12 using TIi DNA polymerase (Promega) or 11 and 13 using Invitrogen Taq DNA polymerase. PCR products were gel-purified using UltraClean DNA purification kits (Mo Bio Laboratories, Inc.), or were enzymatically cleaned using ExoSAP-IT (USB). PCR products were directly sequenced using the SUPAMAC (Sydney University Prince Alfred Macromolecular Analysis Centre) commercial sequencing service on an ABI PRISM® 3700 platform (Applied Biosystems, Foster City), or were sequenced using an ABI BigDyeV3 kit, and run in-house on an ABI PRISM® 3100 platform. The DNA and amino acid (based on nuclear code) TRYP-I sequences obtained from the upstream region, and exons 2 to 8 (as per Table 2) .
  • TGTA(T)A is not recognised by the enzyme.
  • BsrGI PCR-RFLP of TYRP-I Upstream 604 using the restriction enzyme BsrGI for C7 was shown to be heterozygous containing the cut product (123bp) and the uncut product (326bp).
  • C4 is a homozygote for the uncut product (326bp)
  • Cl 7 is a heterozygote for the cut product (123bp) and the uncut product (326bp).
  • the variant G(A)AAAAATTC is recognised and digested by the Xmn I while the variant G(-)AAAAATTC is not recognised by the enzyme.
  • TYRPl Upstream 917, insertion deletion mutation identified via Xmn I PCR-RFLP of TYRP-I Upstream 604 product for C7 was shown to be heterozygous for the cut product (426bp) and the uncut product (149bp).
  • C4 is a homozygote for the cut product (149bp)
  • Cl 7 is a heterozygote for the cut product (149bp) and the uncut product (426bp).
  • the variant A(T)TAAT is recognised and digested by the Ase I while the variant
  • A(C)TAAT is not recognised by the enzyme.
  • Ase I PCR-RFLP of TYRP-I Intron 1 1830 for C7 was shown to be heterozygous for the cut product (347bp) and the uncut product (127bp).
  • C4 is a heterozygote for the cut product (127bp) and the uncut product (347bp)
  • C17 is a homozygote for the cut product (127bp).
  • the variant GG(T)GATAGCCGAC is recognised and digested by the Hph I while the variant GG(C)GATAGCCGAC is not recognised by the enzyme.
  • Hph I PCR-RFLP of TYRP-I Exon 2 2404 after electrophoresis on a 4% agarose gel reflects.
  • C7 as heterozygous for the cut product (339bp) and the uncut product (453bp).
  • C4 is a heterozygote for the cut product (339bp) and the uncut product (453bp)
  • Cl 7 is a homozygote for the uncut product (453bp).
  • the variant GTA(C)G is recognised and digested by the Rsa I while the variant GTA(T)G is not recognised by the enzyme.
  • PCR-RFLP of TYRPl exon 6 using restriction enzyme Rsa I on a 4% agarose gel indicates C7 as heterozygous, containing both the cut allele (158bp) and the uncut allele (186bp).
  • C4 is homozygous for the uncut allele (186bp)
  • C17 is homozygous for the cut allele (158bp).
  • PCR PCR product without digestion
  • DIG PCR product after incubation with the enzyme restriction.
  • TYRPl Exon 8 63 the C/A variation was tested using restriction enzyme Ace II
  • the variant (C)GCG is recognised and digested by the Ace II while the variant (A)GCG is not recognised by the enzyme.
  • PCR-RFLP of TYRP-I exon 8 63 using the restriction enzyme Ace II on a 4% agarose gel indicates C7 to be heterozygous, containing both the cut allele (244bp) and the uncut allele (180bp).
  • C4 is homozygous for the cut allele (180bp)
  • Cl 7 is homozygous for the uncut allele (244bp).
  • Samples C7, C4 and Cl 7 were amplified using PCR conditions described above, with 10 ul of PCR products mixed with 0.5 - 2 units of restriction enzyme, 3 ul of Enzyme Buffer and 7 ul OfH 2 O and incubated for 3 hours at 37 0 C (BstUI at 65 0 C).
  • microsatellite marker for C7 was shown to be heterozygous at position 357, with both 4x 'GA' (l l lbp) and 5x 'GA' repeats (113bp).
  • C4 is a homozygote for 5x 'GA' (113bp) repeat
  • C17 is a homozygote for 4x 'GA' (11 lbp) repeat.
  • insertion deletion mutation marker for C7 was shown to be homozygous for a TCA insertion (179bp). C4 and Cl 7 are heterozygotes, with both the insertion (179bp) and deletion (176bp) fragment lengths. Further, for marker TYRPl Exon 2 2131, insertion deletion mutation marker for C7 was shown to be homozygous for a lbp deletion product size (453bp). C4 is a homozygote for the deletion product size (453bp) while Cl 7 is a heterozygote, with both the 'A' insertion (454bp) and deletion (453bp) fragment lengths.
  • a second indel fragment length (455bp) is also identified by this PCR.
  • C7 was shown to be heterozygous, containing both the cut allele (158bp) and the uncut allele (186bp).
  • C4 is homozygous for the uncut allele (186bp)
  • C17 is homozygous for the cut allele (158bp).
  • PCR PCR product without digestion, DIG - PCR product after incubation with the enzyme restriction.
  • microsatellite marker for C7 was shown to be heterozygous, with product size 300bp and 302bp.
  • C4 is a heterozygote, with product size 295bp and 300bp, while Cl 7 is a heterozygote, with product size 302bp and 302bp.
  • PCR (10 ul) and restriction enzyme digestion conditions as described above were then used to genotype all available Awassi sheep progeny, including the backross mapping population, complex crosses, and other sire families, for seven loci.
  • the families include the following mating designs. In addition to the 177 males of the mapping population, a further 336 animals were generated using the same mating design (backcrossed progeny from mating between a single Awassi/Merino Fl sire (C7) and unselected Merino ewes). Advanced intercrosses were made by crossing C7 to the progeny of three independent Awassi/merino Fl sires (sires A96.0463, A96.0473,
  • Table 14 show summarised genotyping results with the seven loci genotyped, although some loci are currently incompletely genotyped, and secondary (gap filling) genotyping needs to be completed.
  • the TGLA 1C locus was identified as being closest to the estimated QTL location.
  • Progeny were allocated a genotypic score of "1” if the allele passed on by the sire originated from the grandsire, and a genotypic score of "2" if the sire donated an allele originating from the granddam. Phase unknown inheritance was indicated as a "12".
  • the "1" allele was of Awassi origin
  • the "2" allele was of Merino origin.
  • Example 4 Statistical association analysis of TYRP-1 markers and pigmentation Following genotyping of all of the described Awassi families for the seven loci, analysis was conducted to test for an association between known TYRPl haplotypes and PigFibre or ColourScore (1-9). Initially, the same individuals used in the previous analysis (Example 3) were used, with ANOVA results confirming that TYRPl haplotypes ("Combined") explained a large proportion of the variance of the PigFibre trait ( Figure 9, Table 16). Table 16. ANOVA results for PigFibre Trait, with sex, management group and haplotype ("Combined”) as fixed effects

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Abstract

La présente invention concerne un marqueur génétique permettant de distinguer des animaux qui présentent un caractère de pigmentation, ledit marqueur étant un polymorphisme dans le gène de la protéine apparentée à la tyrosinase. L'invention concerne également l'utilisation dudit marqueur pour la prédiction, la sélection, l'amélioration génétique et l'identification d'une pigmentation.
PCT/AU2007/001070 2006-07-31 2007-07-31 Marqueurs de pigmentation WO2008014550A1 (fr)

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KR101109739B1 (ko) 2009-01-08 2012-04-12 사회복지법인 삼성생명공익재단 멜라닌 과다 생성 검출용 마커 및 이의 용도
KR20130016013A (ko) * 2011-08-03 2013-02-14 주식회사 엘지생활건강 미백 피부 타입 유전자 다형성 마커 및 이의 용도
WO2019233557A1 (fr) 2018-06-05 2019-12-12 Wacker Chemie Ag Procédé de revêtement de moyens de soutènement
WO2023001211A1 (fr) * 2021-07-23 2023-01-26 中国农业大学 Puce génétique, combinaison de sondes moléculaires et kit pour l'analyse des caractéristiques de la laine de mouton, et utilisation

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BERALDI D. ET AL.: "Development of a Linkage Map and Mapping of Phenotypic Polymorphism in a Free-living Population of Soay Sheep (Ovis aries)", GENETICS, vol. 173, 2006, pages 1521 - 1537 *
CASTRIANO F. ET AL.: "SUPREME-Project: Sequence of tyrosinase related protein-1 (TR-1) in alpaca", EAAP PUBLICATION, vol. 105, 2001, pages 199 - 206 *
DENG W.D. ET AL.: "Physiological and genetic characteristics of black-boned sheep (Ovis aries)", ANIMAL GENETICS, vol. 37, 2006, pages 586 - 588 *
GRATTEN J. ET AL.: "Compelling evidence that a single nucleotide substitution in TYRP1 is responsible for coat-colour polymorphism in a free-living population of Soay sheep", PROC. R. SOC. B, vol. 274, 2007, pages 619 - 626 *
SMYTH L.M. ET AL.: "Genomic anatomy of the Tyrp1 (brown) deletion complex", PROC. NATL. ACAD. SCI., vol. 103, no. 10, 2006, pages 3704 - 3709 *

Cited By (17)

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Publication number Priority date Publication date Assignee Title
KR101109739B1 (ko) 2009-01-08 2012-04-12 사회복지법인 삼성생명공익재단 멜라닌 과다 생성 검출용 마커 및 이의 용도
KR20130016013A (ko) * 2011-08-03 2013-02-14 주식회사 엘지생활건강 미백 피부 타입 유전자 다형성 마커 및 이의 용도
KR101725556B1 (ko) * 2011-08-03 2017-04-12 주식회사 엘지생활건강 미백 피부 타입 유전자 다형성 마커 및 이의 용도
KR101768423B1 (ko) 2011-08-03 2017-08-16 주식회사 엘지생활건강 미백 피부 타입 유전자 다형성 마커 및 이의 용도
KR101768415B1 (ko) 2011-08-03 2017-08-16 주식회사 엘지생활건강 미백 피부 타입 유전자 다형성 마커 및 이의 용도
KR101768417B1 (ko) 2011-08-03 2017-08-16 주식회사 엘지생활건강 미백 피부 타입 유전자 다형성 마커 및 이의 용도
KR101768418B1 (ko) 2011-08-03 2017-08-16 주식회사 엘지생활건강 미백 피부 타입 유전자 다형성 마커 및 이의 용도
KR101768416B1 (ko) 2011-08-03 2017-08-16 주식회사 엘지생활건강 미백 피부 타입 유전자 다형성 마커 및 이의 용도
KR101768422B1 (ko) 2011-08-03 2017-08-16 주식회사 엘지생활건강 미백 피부 타입 유전자 다형성 마커 및 이의 용도
KR101768419B1 (ko) 2011-08-03 2017-08-16 주식회사 엘지생활건강 미백 피부 타입 유전자 다형성 마커 및 이의 용도
KR101768421B1 (ko) 2011-08-03 2017-08-16 주식회사 엘지생활건강 미백 피부 타입 유전자 다형성 마커 및 이의 용도
KR101768414B1 (ko) 2011-08-03 2017-08-16 주식회사 엘지생활건강 미백 피부 타입 유전자 다형성 마커 및 이의 용도
KR101768424B1 (ko) 2011-08-03 2017-08-16 주식회사 엘지생활건강 미백 피부 타입 유전자 다형성 마커 및 이의 용도
KR101768420B1 (ko) 2011-08-03 2017-08-16 주식회사 엘지생활건강 미백 피부 타입 유전자 다형성 마커 및 이의 용도
KR101768425B1 (ko) 2011-08-03 2017-08-16 주식회사 엘지생활건강 미백 피부 타입 유전자 다형성 마커 및 이의 용도
WO2019233557A1 (fr) 2018-06-05 2019-12-12 Wacker Chemie Ag Procédé de revêtement de moyens de soutènement
WO2023001211A1 (fr) * 2021-07-23 2023-01-26 中国农业大学 Puce génétique, combinaison de sondes moléculaires et kit pour l'analyse des caractéristiques de la laine de mouton, et utilisation

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